Ethanol is among the most widely used drugs, yet the molecular targets to which it binds to produce its effects are not completely understood. Among the strongly-supported protein targets of ethanol are glycine receptors, which in addition to being modulated by alcohol and anesthetics, are also modulated by endogenous zinc. Although the individual effects of zinc and ethanol at the glycine receptor have been studied, the role of endogenous zinc in ethanol enhancement glycine receptor function has not. Our preliminary data indicate that zinc is critical for the action of intoxicating concentrations of ethanol on glycine receptor (GlyR) function, and therefore highlight that understanding zinc/ethanol interactions is necessary in defining the mechanism of action of ethanol at the GlyR. Subsequently, understanding zinc/ethanol interactions could lead to more accurate approximations of ethanol's effects in vivo. The proposed project will investigate the effects of zinc on alcohol action at the glycine receptor using in vitro and vivo techniques. Aim 1 will test and characterize a zinc/ethanol interaction in three glycine receptor subunits (11, 1 2, and 13) expressed in Xenopus oocytes. Aim 2 will determine a mechanism for the zinc-ethanol interaction characterized in Aim 1 using site-directed mutagenesis to eliminate putative zinc binding sites on the glycine receptor. Mutant glycine receptors will be constructed based on amino acid residues known to be important for zinc binding and will allow for it to be determined if zinc/ethanol interactions at the glycine receptor are due to zinc binding at known sites or via action at alternative sites. The mutations will be initially created in the 1 1 subunit, but if significant results are obtained, then additional mutations in the homologous positions in the 1 2 and 1 3 subunits will be made. Aim 3 will evaluate the effects of a zinc-insensitive glycine receptor mutation on alcohol consumption and other behavioral tests in mice. Homozygous Glra1(D80A) mice, which contain a mutation at a putative 1 1 glycine receptor zinc binding site, will provide an animal mode for behavioral studies of zinc/ethanol interactions at the glycine receptor, and will be potentially the first glycine receptor homozygous knock-in mice in which alcohol consumption and other behavioral measures of ethanol action will be tested. Unlike other homozygous glycine receptor KI mice, which have not been viable, Glra1(D80A) homozygotes are viable as adults. Findings from this project will provide insight about the effects of zinc on alcohol's action in the central nervous system. By better understanding the sites and mechanisms of action of alcohol, more effective treatments for alcohol abuse and alcoholism can be developed. ) PUBLIC HEALTH RELEVANCE: Ethanol is among the most widely used drugs, yet the molecular targets to which it binds to produce its effects are not completely understood. By better understanding the sites and mechanisms of action of alcohol, more effective treatments for alcohol abuse and alcoholism can be developed.